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In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
The essential Ubc4/Ubc5 function in yeast is HECT E3-dependent, and RING E3-dependent pathways require only
Kate E Stoll1, Peter S Brzovic, Trisha N Davis
1Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
The Journal of Biological Chemistry
|March 2, 2011
Summary
The yeast ubiquitin-conjugating enzymes Ubc4 and Ubc5 are essential for cell viability. Their redundant function is primarily with the HECT-type E3 ligase Rsp5, acting as monoubiquitinating enzymes in RING E3 pathways.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Ubc4 and Ubc5 are ubiquitin (Ub)-conjugating enzymes crucial for various ubiquitination pathways in yeast.
- These pathways include those mediated by Rsp5 and the anaphase-promoting complex (APC).
- The double deletion of UBC4 and UBC5 genes in yeast is lethal, indicating their essential, redundant function.
Purpose of the Study:
- To investigate the essential pathway disrupted by the combined deletion of UBC4 and UBC5.
- To elucidate the specific roles of Ubc4 and Ubc5 in essential ubiquitination processes.
- To differentiate the functions of Ubc4/Ubc5 in HECT-type versus RING E3 ligase pathways.
Main Methods:
- Introduction of point mutations into the Ubc4 gene to assess functional rescue of the lethal ubc4/ubc5 deletion phenotype.
- Analysis of mutations affecting the active site, E3-binding, RING E3-catalyzed isopeptide formation, HECT E3 transthiolation, and Ub-binding.
- Comparison of Ubc4/Ubc5 function with other ubiquitin-conjugating enzymes like Ubc1 in APC-mediated ubiquitination.
Main Results:
- Ubc4 active site and E3-binding mutations failed to rescue the lethal phenotype, highlighting the requirement for an active E3/E2∼Ub complex.
- A Ubc4 mutation (N78S) specifically eliminating RING E3-catalyzed isopeptide formation rescued the lethality, implicating HECT E3 ligases.
- The essential redundant function of Ubc4/Ubc5 is with the HECT-type E3 ligase Rsp5, likely the sole essential HECT in yeast.
- Ubc1 showed weak ability to replace Ubc4 for monoubiquitination with APC, but Ubc4 could not replace Ubc1 for polyubiquitination chain extension.
- A backside Ub-binding mutant of Ubc4 (S23R) had no observable effect.
Conclusions:
- Ubc4 and Ubc5 are the primary E2 enzymes for Rsp5 in yeast.
- Ubc4 and Ubc5 function as monoubiquitinating E2s in RING E3-catalyzed pathways, distinct from their human ortholog UbcH5.
- These findings clarify the essential, redundant roles of Ubc4 and Ubc5 in yeast ubiquitination.
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